The enzyme, thymidylate synthase (TS) catalyzes the reaction that produces thymidylate (TMP). Depletion of TMP by inhibition of TS results in death of rapidly dividing cells, such as cancer cells. TS has been the focus of extensive research in cancer experimental therapeutics and is a well-validated target. TS inhibitors in current clinical use (e.g. 5-fluorouracil and raltitrexed) are limited in their utility by drug resistance. A main factor that is thought to underlie clinical drug resistance is an elevation of TS levels, which may be an intrinsic property of the tumor or may be induced by treatment with the current TS inhibitors. Human TS (hTS) exists in 2 conformations: 1 active and 1 inactive, which are in apparent equilibrium. We propose to test a hypothesis that TS inhibition via stabilization of the inactive conformer will not only prevent the induction in TS levels that is observed with classical active site directed inhibitors but also reduce TS levels in tumors with elevated expression of TS. To achieve this goal, mutant hTSs that are stabilized in either the active or inactive conformation will be created and characterized. These mutants will be utilized to analyze the effect of stabilization in an inactive conformation on the intracellular stability of the protein. Also, small molecule ligands stabilizing the inactive conformation will be developed. If our hypothesis is correct, then there is a strong rationale for developing inhibitors that would work by stabilization of the inactive conformation as opposed to current inhibitors that stabilize the active conformation. Such inhibitors should not only inhibit the catalytic activity of TS but also work by reducing the levels of TS protein, eliminating a major mechanism of resistance. We also propose to utilize hTS as a model for studies of synergistic inhibition by a mixture of inhibitors. This novel approach, which we term hetero-inhibition, would capitalize on the asymmetry of the hTS homodimer and may have general utility for the inhibition of proteins that are functional and/or structural heterodimers.